Methods to produce high levels of C. difficile toxins

ABSTRACT

The present invention relates to the field of medical immunology and further to pharmaceutical compositions, methods of making and methods of use of vaccines. More specifically this invention relates to recombinant proteins derived from the genes encoding  Clostridium difficile  toxin A and toxin B, and their use in an active vaccine against  C. difficile.

TECHNICAL FIELD OF INVENTION

[0001] The present invention relates to the field of medical immunologyand further to pharmaceutical compositions, methods of making andmethods of use of vaccines. More specifically this invention relates torecombinant proteins derived from the genes encoding Clostridiumdifficile toxin A and toxin B, and their use in an active vaccineagainst C. difficile.

BACKGROUND OF THE INVENTION

[0002]Clostridium difficile, a Gram positive anaerobic spore-formingbacillus is an etiologic agent of antibiotic associated diarrhea (AAD)and colitis (AAC). The symptoms of the disease range from mild diarrheato fulminant and life-threatening pseudomembranous colitis (PMC).Antibiotic therapy can disrupt the normal intestinal microflora.Destruction of the normal flora results in a condition in which C.difficile can spores of C. difficile can germinate and the organism cangrow and produce disease causing toxins. C. difficile causes about 25%of antibiotic-associated diarrheas, however, it is almost always thecausative agent of PMC (Lyerly, D. M. and T. D. Wilkins, in Infectionsof the Gastrointestinal Tract, Chapter 58, pages 867-891, (Raven Press,Ltd, New York 1995)). Additionally, C. difficile is frequentlyidentified as a causative agent of nosocomial infectious diarrheas,particularly in older or immuno-compromised patients (U.S. Pat. No.4,863,852 (Wilkins et al.) (1989)).

[0003] Disease caused by C. difficile is due to two enteric toxins A andB produced by toxigenic strains (U.S. Pat. No. 5,098,826 (Wilkins etal.) (1992)). Toxin A is an enterotoxin with minimal cytotoxic activity,whereas toxin B is a potent cytotoxin but has limited enterotoxicactivity. The extensive damage to the intestinal mucosa is attributableto the action of toxin A, however, toxins A and B act synergistically inthe intestine.

[0004] The genetic sequences encoding both toxigenic proteins A and B,the largest known bacterial toxins, with molecular weights of 308,000and 269,000, respectively, have been elucidated (Moncrief et al.,Infect. Immun. 65:1105-1108 (1997); Barroso et al., Nucl. Acids Res.18:4004 (1990); Dove et al. Infect. Immun. 58:480-488 (1990)). Becauseof the degree of similarity when conserved substitutions are considered,these toxins are thought to have arisen from gene duplication. Theproteins share a number of similar structural features with one another.For example, both proteins possess a putative nucleotide binding site, acentral hydrophobic region, four conserved cysteines and a long seriesof repeating units at their carboxyl ends. The repeating units of toxinA, particularly, are immunodominant and are responsible for binding totype 2 core carbohydrate antigens on the surface of the intestinalepithelium (Krivan et al., Infect. Immun. 53:573-581 (1986); Tucker, K.and T. D. Wilkins, Infect. Immun. 59:73-78 (1991)).

[0005] The toxins share a similar molecular mechanism of actioninvolving the covalent modification of Rho proteins. Rho proteins aresmall molecular weight effector proteins that have a number of cellularfunctions including maintaining the organization of the cytoskeleton.The covalent modification of Rho proteins is due to glucosyltransferaseactivity of the toxins. A glucose moiety is added to Rho usingUDP-glucose as a co-substrate (Just et al. Nature 375:500-503 (1995),Just et al. J. Biol. Chem 270:13932-13939 (1995)). Theglucosyltransferase activity has been localized to approximately theinitial 25% of the amino acid sequence of each of these toxins (Hofmannet al. J. Biol. Chem. 272:11074-11078 (1997), Faust and Song, Biochem.Biophys. Res. Commun. 251:100-105 (1998)) leaving a large portion of thetoxins, including the repeating units, that do not participate in theenzymatic activity responsible for cytotoxicity.

[0006] The toxin A protein comprises 31 contiguous repeating units (rARU) and may contain multiple T cell epitopes (Dove et al. Infect. Immun.58:480-488 (1990). The repeating units are defined as class I repeatsand class II. rARU may be uniquely suited for use in inducing Tcell-dependent response to an antigen. The sequence of each unit issimilar but not identical. These features along with its usefulness ineliciting toxin A neutralizing antibodies make rARU a novel candidate asa carrier protein.

[0007] The toxin B repeating units have similar features to those ofrARU. Like rARU, the recombinant toxin B repeating units (rBRU) arerelatively large (˜70 kDa) and are composed of contiguous repeats ofsimilar amino acid sequences (Barroso et al. Nucleic Acids Res. 18:4004(1990); Eichel-Streiber et al. Gene 96:107-113 (1992)). Less is knownabout this portion of toxin B than the binding domain of toxin A.

[0008] Thomas et al (U.S. Pat. No. 5,919,463 (1999)) disclose C.difficile toxin A or toxin B or certain fragments thereof as mucosaladjuvants intranasally administered to stimulate an immune response toan antigen (e.g., Helicobacter pylori urease, ovalbumin (OVA), orkeyhole limpet hemocyanin (KLH)). However, Thomas does not teach the useof such adjuvant for protection against strains of C. difficile. Lyerlyet al. Current Microbiology 21:29-32 (1990) considered at a smallerrecombinant fragment from the toxin A repeats in hamster protectionassays. However, these data suggest at best only a very weak or partialprotection from strains of C. difficile, whereas the present inventiondemonstrates the use of C. difficile toxin repeating units that providea clear immunogenic response and at higher levels, which affordprotection against C. difficile.

[0009] Even were one to consider rARU and rBRU as candidate proteins forconjugate vaccines, the production of such proteins presents certainchallenges. There are methods for the production of toxin A andantibodies elicited thereto (U.S. Pat. No. 4,530,833 (Wilkins et al.)(1985); U.S. Pat. No. 4,533,630 (Wilkins et al.) (1985); and U.S. Pat.No. 4,879,218 (Wilkins et al.) (1989)). There are significantdifficulties in producing sufficient quantities of the C. difficiletoxin A and toxin B proteins. These methods are generally cumbersome andexpensive. However, the present invention provides for the constructionand recombinant expression of a nontoxic truncated portions or fragmentsof C. difficile toxin A and toxin B in strains of E. coli. Such methodsare more effective and commercially feasible for the production ofsufficient quantities of a protein molecule for raising humoralimmunogenicity to antigens.

[0010] Part of the difficulty that the present invention overcomesconcerns the fact that large proteins are difficult to express at highlevels in E. coli. Further, an unusually high content of AT in theseclostridial gene sequences (i.e., AT-rich) makes them particularlydifficult to express at high levels (Makoffet al. Bio/Technology7:1043-1046 (1989)). It has been reported that expression difficultiesare often encountered when large (i.e., greater than 100 kd) fragmentsare expressed in E. coli. A number of expression constructs containingsmaller fragments of the toxin A gene have been constructed, todetermine if small regions of the gene can be expressed to high levelswithout extensive protein degradation. In all cases, it was reportedthat higher levels of intact, full length fusion proteins were observedrather than the larger recombinant fragments (Kink et al., U.S. Pat. No.5,736,139; see: Example 11(c)). It has been further reported thatAT-rich genes contain rare codons that are thought to interfere withtheir high-level expression in E. coli (Makoff et al. Nucleic AcidsResearch 17:10191-10202). The present invention provides for methods toproduce genes that are both large and AT-rich and immunogeniccompositions thereof. For example, the toxin A repeating units areapproximately 98 kDa and the gene sequence has an AT content ofapproximately 70% that is far above the approximately 50% AT content ofthe E. coli geneome. The present invention provides for methods ofexpressing AT-rich genes (including very large ones) at high levels inE. coli without changing the rare codons or supplying rare tRNA.

[0011] Citation of the above documents is not intended as an admissionthat any of the foregoing is pertinent prior art. All statements as tothe date or representation as to the contents of these documents isbased on the information available to the applicants and does notconstitute any admission as to the correctness of the dates or contentsof these documents. Further, all documents referred to throughout thisapplication are incorporated in their entirety by reference herein.Specifically, the present application claims benefit of priority to U.S.provisional patent application serial No. 60/190,111, which was filed onMar. 20, 2000; and U.S. provisional patent application serial No.60/186,201, which was filed on Mar. 1, 2000; and U.S. provisional patentapplication serial No. 60/128,686, which was filed on Apr. 9, 1999, andwhich provisional patent applications are incorporated in their entiretyby reference herein.

SUMMARY OF THE INVENTION

[0012] The present invention is drawn to an immunogenic composition thatincludes recombinant proteins. The genes encoding the proteins areisolated from a strain of C. difficile. A preferred embodiment of thisinvention provides that at least one protein is a toxin or a toxinfragment. A further preferred embodiment provides that the toxin is C.difficile toxin A or toxin B. A more preferred embodiment of the presentinvention provides that the recombinant protein components are nontoxicand comprise a portion of both toxins including all of the amino acidsequence of the C. difficile toxin A or toxin B repeating units (rARU orrBRU) or fragment thereof. The immunogenic composition may furtherinclude a carbohydrate moiety as well as a pharmaceutically acceptablecarrier or other compositions in a formulation suitable for injection ina mammal.

[0013] Another embodiment of the invention is that the rARU and rBRUcomponents are combined, preferably in a manner that results in highlevels of neutralizing antibodies to toxins A and B when the immunogeniccomposition is used in vaccine. The components may be admixed atdifferent ratios. Further, the rARU and rBRU components may bechemically or physically linked to form a complex. Another preferredembodiment is that the rARU and rBRU sequences, or fragments thereof,may be genetically fused in a manner that results in the production of ahybrid molecule. A further embodiment is that the immunogeniccomposition elicits antibodies that precipitate the native C. difficiletoxins and neutralize their cytotoxic activity thus providing protectionagainst C. difficile associated disease.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014]FIG. 1. Schematic of toxins A and B. The repeating units of thetoxins function in binding to the cell surface. Antibodies to therepeating units of the toxins neutralize cytotoxic activity by blockingthe binding of the toxins to the cell surface.

[0015]FIG. 2 shows the nucleotide sequence (numbers 5690-8293, GenBankaccession number M30307, Dove et al. 1993) of the toxin A gene regionthat encodes rARU and the toxin A stop codon. The sequence encodes forthe entire repeating units of toxin A from C. difficile strain VPI 10463as defined by Dove et al. (Dove et al., Infect Immun. 58:480-488(1990)). In addition it encodes for 4 amino acids upstream of thebeginning of the repeating units and a small stretch of hydrophobicamino acids at the end of toxin A. The Sau3A site (underlined) at thebeginning of the sequence was used to subclone the gene fragment to anexpression vector. The stop codon at the end of the sequence isitalicized.

[0016]FIG. 3 shows the amino acid sequence (GenBank accession numberM303307) of rARU. The invention contemplates the use of any recombinantprotein containing this amino acid sequence, any fragment therein, anyfusion protein containing rARU or a fragment therein, and any largerfragment from toxin A carrying all or part of rARU, as a carrier forconjugate vaccine compositions.

[0017]FIG. 4 shows the expression vector pRSETB-ARU-Km^(r) used forexpression of rARU. A Sau3A/HindIII gene fragment of approximately 2.7kb containing the entire nucleotide sequence encoding rARU, stop codon,and a small region downstream of the toxin A stop codon, was subclonedto the vector pRSETB digested with BamHI and HindIII. In a subsequentstep the kanamycin resistance gene was subcloned at the HindIII sitelocated downstream of the rARU gene fragment. The 1.2 kb fragmentencoding the Km^(r) gene was derived from pUC4K (GenBank accessionnumber X06404) by digestion with EcoRI and subcloned at the HindIII siteafter blunt ending of the vector and Km^(r) cassette with Klenowfragment. Expression vector pRSETB-ARU-Km^(r) was transformed intoBL21(DE3) for expression of rARU under control of the T7 promoter.

[0018] * HindIII/EcoRI sites were eliminated by blunt ending.

[0019]FIG. 5 shows an SDS-PAGE gel (15% acrylamide) of rARU expressionand purification steps. Lanes: 1) 4 μl of 10× BL21(DE3) E.coli/pRSETB-ARU-Km^(r) lysate 2) 4 μl of dialyzed 40% ammonium sulfatefraction at 10× relative to the original culture volume 3) 5 μl rARU(0.88 mg/ml) purified by CL-6B Sepharose anion exchange chromatography.

[0020]FIG. 6 shows the nucleotide sequence (GenBank accession numberX531138, Wilkins et al. 1990) of the toxin B gene region that encodesrBRU and a small portion upstream. The Sau3a restriction sites used forsubcloning are underlined. The sequence of the repeating units of toxinB from C. difficile strain VPI was defined previously (Eichel-Streiberet al. Mol. Gen. Gen. 233:260-268).

[0021]FIG. 7 shows the amino acid sequence (GenBank accession numberX53138) of rBRU and a small upstream region. The invention contemplatesthe use of any recombinant protein containing this amino acid sequence,any fragment therein, any fusion protein containing rBRU or a fragmenttherein, and any larger fragment from toxin B carrying all or part ofrBRU, as a component in a vaccine against C. difficile.

[0022]FIG. 8 shows the expression vector pRSETC-BRU-Km^(r) used forexpression of rBRU. A gene fragment of approximately 1.8 kb containingnearly the entire nucleotide sequence encoding rBRU (final 10 aminoacids of toxin B are eliminated) was subcloned from the toxin B gene(Phelps et al. Infect. Immun. 59:150-153 (1991)) to pGEX-3X. ABamHI/EcoRI from pGEX-3X-BRU was subcloned to pRSETC. In a subsequentstep the kanamycin resistance gene was subcloned at the EcoRI sitelocated downstream of the rBRU gene fragment. The 1.2 kb fragmentencoding the Km^(r) gene was derived from pUC4K (GenBank accessionnumber X06404) by digestion with EcoRI. Expression vectorpRSETC-BRU-Km^(r) was transformed into BL21 (DE3) for expression ofrBRUunder control of the T7 promoter.

[0023]FIG. 9. SDS-PAGE of purified rARU and partially purified rBRU.Lanes; 1) rARU purified by sequential ammonium sulfate precipitation andSepharose CL-6B anion exchange chromatography, 2) rBRU partiallypurified by ammonium sulfate precipitation and hydrophobic interactionchromatography on phenyl Sepharose, 3) lysate (10× concentration) ofEscherichia coli BL21 (DE3)/pRSETC-BRU-Km^(r).

[0024]FIG. 10. Crossed-immunoelectrophoresis of (A) C. difficile culturefiltrate and (B) partially purified rBRU. C. difficile goat antisera wasused as the precipitating antibody.

[0025]FIG. 11. shows an example of a genetic fusion of rARU and rBRU. ASau3A/EcoRI toxin A gene fragment (nucleotides 5530 through 6577) may befused to an ApoI toxin B gene fragment (nucleotides 5464 through 6180)to create an in-frame 1,763 bp gene fusion expressing a 588 amino acidrARU'/'rBRU' fusion protein of approximately 68 kDa containing asignificant portion of the repeating units from both toxin genes. TherARU' fragment encodes an epitope for PCG-4 represented by the open barin the rARU' portion of the gene fusion.

DETAILED DESCRIPTION OF THE INVENTION

[0026] The present invention is drawn to an immunogenic composition thatincludes at least one recombinant protein component, wherein the geneencoding the protein component is isolated from a strain of Clostridiumdifficile. A preferred embodiment of this invention provides that theprotein is a toxin or a toxin fragment. An even further preferredembodiment provides that the toxin is toxin A, with yet a furtherpreferred embodiment being a portion of the toxin containing all of theamino acid sequence of the toxin A repeating units (rARU) or fragmentthereof. Another preferred embodiment is that the toxin is toxin B, withyet another preferred embodiment being a portion of the toxin containingall of the amino acid sequence of the repeating units (rBRU) or afragment thereof. The immunogenic composition may further include apharmaceutically acceptable carrier or other compositions in aformulation suitable for injection in a mammal.

[0027] These immunogenic compositions of the present invention elicit animmune response in a mammalian host, including humans and other animals.The immune response may be either a cellular dependent response or anantibody dependent response or both and further the response may provideimmunological memory or a booster effect or both in the mammalian host.These immunogenic compositions are useful as vaccines and may provide aprotective response by the mammalian subject or host to infection bystrains of Clostridium difficile.

[0028] The present invention further includes methods for producing animmunogenic composition by: constructing a genetic sequence encoding arecombinant protein component, where the gene encoding the proteincomponent is isolated from a strain of Clostridium difficile, expressingthe recombinant protein component in a microbial host; recovering therecombinant protein from a culture of the host; conjugating the proteinto a second protein component, and recovering the conjugated protein andpolysaccharide component. The protein component may also consist of afusion protein, whereby a portion of said recombinant protein isgenetically fused to a second protein component. Preferably theexpression of the genetic sequence is regulated by an inducible promoterthat is operatively positioned upstream of the sequence and isfunctional in the host. Even further, said genetic sequence ismaintained throughout the growth of the host by constant and stableselective pressure. Maintenance of the expression vector may beconferred by incorporation in the expression vector of a geneticsequence that encodes a selective genotype, the expression of which inthe microbial host cell results in a selective phenotype. Such selectivegenotypes, include a gene encoding resistance to antibiotics, such askanamycin. The expression of this selective genotypic sequence on theexpression vector in the presence of a selective agent or condition,such as the presence of kanamycin, results in stable maintenance of thevector throughout growth of the host. A selective genotype sequencecould also include a gene complementing a conditional lethal mutation.

[0029] Other genetic sequences may be incorporated in the expressionvector, such as other drug resistance genes or genes that complementlethal mutations.

[0030] Microbial hosts of this invention may include: Gram positivebacteria; Gram negative bacteria, preferably E. coli; yeasts;filamentous fungi; mammalian cells; insect cells; or plant cells.

[0031] The methods of the present invention also provide for a level ofexpression of the recombinant protein in the host at a level greaterthan about 10 mg/liter of the culture, more preferably greater thanabout 50 mg/liter and even more preferably at 100 mg/liter or greaterthan about 100 mg/liter. The molecular weight of the protein is greaterthan about 30 kDa, preferably greater than about 50 kDa and even morepreferably greater than about 90 kDa. This invention also provides thatthe protein may be recovered by any number of methods known to those inthe art for the isolation and recovery of proteins, but preferably therecovery is by ammonium sulfate precipitation followed by ion exchangechromatography.

[0032] The present invention further includes methods for preparing theimmunogenic composition that provides that the protein component isconjugated to a second protein component by one of a number of meansknown to those in the art, particularly an amidization reaction.

[0033] Also, high yields of recombinant protein may be dependent on thegrowth conditions, the rate of expression, and the length of time usedto express AT-rich gene sequences. In general, AT-rich genes appear tobe expressed at a higher level in E. coli during a post-exponential orslowed phase of growth. High-level production of the encoded proteinrequires moderate levels of expression over an extended period (e.g.20-24 h) of post-exponential growth rather than the typical approach ofhigh-level expression during exponential growth for shorter periods(e.g. 4-6 h). In this regard, it is more efficient to maintain plasmidscarrying the gene of interest by maintaining constant selective pressurefor the gene or its expression vector during the extended period ofgrowth. One aspect of the present invention is using an antibiotic thatis not inactivated or degraded during growth of the expression host cellas is found with ampicillin. One such preferred embodiment involves theexpression of genes encoding resistance to kanamycin as the selectivephenotype for maintaining the expression vector which comprises suchkanamycin resistance genetic sequences. Expression of large AT-richclostridial genes in E. coli at levels (>100 mg/liter) provided for bymethods of the present invention was hitherto unknown.

[0034] Terms as used herein are based upon their art recognized meaningand should be clearly understood by the ordinary skilled artisan.

[0035] An immunogenic composition is any composition of material thatelicits an immune response in a mammalian host when the immunogeniccomposition is injected or otherwise introduced. The immune response maybe humoral, cellular, or both.

[0036] A fusion protein is a recombinant protein encoded by a gene orfragment of a gene, genetically fused to another gene or fragment of agene.

[0037] A booster effect refers to an increased immune response to animmunogenic composition upon subsequent exposure of the mammalian hostto the same immunogenic composition. A humoral response results in theproduction of antibodies by the mammalian host upon exposure to theimmunogenic composition.

[0038] rARU is a recombinant protein containing the repeating units ofClostridium difficile toxin A as defined by Dove et al. (Dove et al.Infect. Immun. 58:480-488 (1990)). The nucleotide sequence encoding rARUand the amino acid sequence of rARU are shown in FIGS. 2 and 3,respectively. The rARU expressed by pRSETB-ARU-Km^(r) contains theentire repeating units region of toxin A. The invention furthercontemplates the use of this recombinant protein component, or any otherprotein component containing the entire repeating units of toxin A orany fragment therein, whether expressed alone or as a fusion protein.

[0039] Similar methods may be used to isolate, clone and express arecombinant protein component comprising the repeating units ofClostridium difficile toxin B (rBRU). The nucleotide sequence encodingrBRU and the amino acid sequence of rBRU are shown in FIGS. 6 and 7,respectively. The rBRU expressed by pRSETC-BRU-Km^(r) contains theentire repeating units region of toxin B (see FIG. 8).

[0040] The present methods provide for preparation of immunogeniccompositions comprising rARU or rBRU or both, which are useful asvaccines. Immunogenic compositions may be formulated as vaccines in apharmaceutically acceptable carrier or diluent (e.g., water, a salinesolution (e.g., phosphate-buffered saline), a bicarbonate solution(e.g., 0.24 M NaHCO.sub.3), a suppository, cream, or jelly), which areselected on the basis of the mode and route of administration, andstandard pharmaceutical practice, see: U.S. Pat. No. 5,919,463 Thomas,et al., (1999), which is incorporated in its entirety by referenceherein. Suitable pharmaceutical carriers and diluents, as well aspharmaceutical necessities for their use in pharmaceutical formulations,are described in Remington's Pharmaceutical Sciences (Alfonso Gennaro etal., eds., 17th edn., Mack Publishing Co., Easton Pa., 1985), a standardreference text in this field, in the USP/NF, and by Lachman et al. (TheTheory & Practice of Industrial Pharmacy, 2nd edn., Lea & Febiger,Philadelphia Pa., 1976). In the case of rectal and vaginaladministration, the vaccines are administered using methods and carriersstandardly used in administering pharmaceutical materials to theseregions. For example, suppositories, creams (e.g., cocoa butter), orjellies, as well as standard vaginal applicators, droppers, syringes, orenemas may be used, as determined to be appropriate by one skilled inthe art.

[0041] The vaccine compositions of the invention may be administered byany route clinically indicated, such as by application to the surface ofmucosal membranes (including: intranasal, oral, ocular,gastrointestinal, rectal, vaginal, or genito-urinary). Alternatively,parenteral (e.g., intravenous, subcutaneous, intraperitoneal, orintramuscular) modes of administration may also be used. The amounts ofvaccine administered depend on the particular vaccine antigen and anyadjuvant employed; the mode and frequency of administration; and thedesired effect (e.g., protection and/or treatment), as determined by oneskilled in the art. In general, the vaccines of the invention will beadministered in amounts ranging between 1 μg and 100 mg. Administrationis repeated as is determined to be necessary by one skilled in the art.For example, a priming dose may be followed by 3 booster doses at weeklyintervals.

[0042] Having now generally described the invention, the same will bemore readily understood through reference to the following exampleswhich are provided by way of illustration, and are not intended to belimiting of the present invention, unless specified.

EXAMPLES Example 1

[0043] Construction of rARU Expression Vector.

[0044] The vector pRSETB-ARU-Km^(r) used for expression and purificationwas constructed using standard techniques for cloning (Sambrook et al.,Molecular Cloning: A Laboratory Manual (1989)). The nucleotide sequenceof the toxin A gene fragment encoding rARU was derived from the clonedtoxin A gene (Dove et al., Infect. Immun. 58:480-488 (1990); Phelps etal., Infect Immun. 59:150-153 (1991)) and is shown in FIG. 2. The genefragment encodes a protein 867 amino acids in length (FIG. 3) with acalculated molecular weight of 98 kDa. The gene fragment was subclonedto the expression vector pRSETB. A kanamycin resistance gene wassubsequently subcloned to the vector. The resulting vectorpRSETB-ARU-Km^(r) expresses rARU. An additional 31 amino acids at theN-terminus of the recombinant protein are contributed by the expressionvector pRSETB. The final calculated molecular weight of the recombinantprotein is 102 kDa.

Example 2

[0045] Expression and Purification of rARU.

[0046]Escherichia coli T7 expression host strain BL21(DE3) wastransformed with pRSETB-ARU-Km^(r) as described (Sambrook et al.Molecular Cloning: A Laboratory Manual (1989)). One liter cultures wereinoculated with 10 ml of overnight growth of Escherichia coli BL21(DE3)containing pRSETB-ARU-Km^(r) and grown at 37° C. in Terrific broth(Sigma, St. Louis, Mo.) containing 25 μg/ml of kanamycin to an O.D. 600of 1.8-2.0 and isopropyl B-D-thiogalactopyranoside (IPTG) was added to afinal concentration of 40 μM. Cells were harvested after 22 h ofinduction, suspended in 0.1 liter of standard phosphate buffered saline,pH 7.4, containing 0.2% casamino acids, and disrupted by sonication.Cellular debris was removed from the lysate by centrifugation. Lysatestypically contained a titer (reciprocal of the highest dilution with anA₄₅₀ greater than 0.2) of 10⁶ in the TOX-A test EIA (TechLab, Inc.,Blacksburg, Va.). Lysates were saturated with 40% ammonium sulfate,stirred at 4° C. overnight and precipitating proteins were harvested bycentrifugation. The ammonium sulfate fraction was suspended in 0.1liters of 5 mM K₂PO₄, 0.1 M NaCl₂, pH 8.0 and dialyzed extensivelyagainst the same buffer at 4° C. Insoluble material was removed bycentrifugation. The dialyzed solution was passed through a columncontaining Sepharose CL-6B chromatography media (50 ml media/100 mlsolution). Fractions were collected and monitored for the presence ofrARU by EIA using the TOX-A test. Fractions containing EIA activity wereanalyzed by SDS-PAGE for the presence of rARU at a molecular weight ofapproximately 102 kDa. Fractions containing a single band of rARU werepooled. To further ensure purity the pooled solution was again passedover a Sepharose CL-6B column (25 ml media/100 ml protein solution). Thesolution containing purified rARU was filtered sterilized by passagethrough a 22μ filter and stored at 4° C. Purified rARU along withsamples from the steps of purification (lysate and dialyzed ammoniumsulfate fraction) are shown in FIG. 5. The procedure typically yieldsapproximately 100 mg rARU per liter of E. coli/pRSETB-ARU-Km^(r)culture. A combined 6-liter batch yielded 0.850 liters of rARU at 0.88mg/ml for a total of 748 mg of rARU or 125 mg/liter of culture. Theamount of rARU recovered represented 23% of the total soluble protein.

Example 3

[0047] Construction of rBRU Expression Vector.

[0048] The vector pRSETC-BRU-Km^(r) used for expression and purificationwas constructed using standard techniques for cloning (Sambrook et al.,Molecular Cloning: A Laboratory Manual (1989)). The nucleotide sequenceof the toxin B gene fragment encoding rBRU was derived from the clonedtoxin B gene (Barroso et al., Nucleic Acids Res 18:4004 (1990)) and isshown in FIG. 6. The gene fragment encodes a protein 622 amino acids inlength with a molecular weight of approximately 70 kDa. The genefragment was subcloned to the expression vector pRSETC. A kanamycinresistance gene was subsequently subcloned to the vector. The resultingvector pRSETC-BRU-Km^(r) expresses rBRU.

Example 4

[0049] High-level Expression and Partial Purification of rBRU.

[0050] One liter of Escherichia coli pRSETC-BRU-Km^(r) was grown for 25h at 37° C. in a shaking incubator. Cells were harvested bycentrifugation and resuspended in 0.1 liter phosphate buffered salinewith 0.2% casamino acids. Supernatant of the culture at harvest had a pHof 6.2. Cells were disrupted by sonication and cellular debri wasremoved by centrifugation. The 10× lysate is shown in FIG. 9, lane 3.

Example 5

[0051] Immune response to the rARU component of the conjugates.

[0052] Antibodies to C. dfficile toxin A (CDTA). Antibodies to nativetoxin A were measured by ELISA, with toxin A isolated from C. difficileas the coating antigen, and by in-vitro neutralization of cytotoxicity(Lyerly et al. Infect. Immun. 35:1147-1150 (1982)). Human intestinalepithelial HT-29 cells (ATCC HTB 38) were maintained in 96 well plateswith McCoy's 5A medium supplemented with 10% fetal calf serum in a 5%CO₂ atmosphere. HT-29 cells were chosen because of their highsensitivity to CDTA probably because of the high density of thecarbohydrate receptor on their surface. Serial 2-fold dilutions of serawere incubated with 0.4 μg/ml of CDTA for 30 min at room temperature.CDTA-serum mixtures were added to the wells at a final concentration of20 ng of toxin A per well (about 200 times the minimal cytotoxic dosefor HT-29 cells) in a final volume of 0.2 ml. The neutralization titeris expressed as the reciprocal of the TABLE 1 Serum antibodies (μg/ml)to Clostridium difficile toxin A (CDTA) elicited in mice by recombinantenterotoxin A (rARU) or polysaccharides bound to rARU alone orsuccinylated (rARUsucc) μg ELISA (Geometric mean and 25-75 centiles)rARU First Conjugate Injected injection Second injection Third injectionrARU* 6.94 ND ND 717 (621-863) Pn14-rARU 1.29  3.70 80.1 (69.8-131) 194(113-236) (2.55-5.08) Pn14rARU 7.30  7.94  183 (146-175) 371 (274-463)succ (5.21-11.3) SF-rARU 3.90 ND  433 (258-609) 613 (485-778) SF-rARU6.94 ND  191 (118-291) 518 (366-615) succ SF-rARU* 3.90 ND ND 437(372-547) SF-rARU 6.94 ND ND 242 (172-443) succ* K1 8.08 10.7 84.9(72.5-131) 390 (279-470) (6.75-17.2)

[0053] highest dilution that completely neutralized cytotoxicity.

[0054] All 5 conjugates elicited high levels of anti-CDTA (194-613μg/ml) (Table 1). Since the 2.5 μg immunizing dose of the conjugates wasbased on its polysaccharide content, the amount of rARU injected wasdifferent for each conjugate. For example, on a protein weight basis,Pn14-rARU, with 1.29 μg of rARU, elicited 194 μg CDTA antibody/ml (150.3μg Ab/μg rARU injected). In contrast, Pn14-rARUsucc, that contained 7.3μg of rARU per dose, elicited 371 μg CDTA antibody/ml (50.8 μg Ab/μgrARUsucc injected). Pn14-rARU induced more anti-CDTA per μg rARU thanPn14-rARUsucc, however, the total amount of anti-CDTA elicited byPn14-rARUsucc was greater due to its higher content of rARU. Thedifference between the levels of anti-CDTA elicited by Pn14-rARU (194 μgCDTA antibody/ml) compared with Pn14-rARUsucc (371 μg CDTA antibody/ml)was significant.

[0055] SF-rARU, containing 3.9 μg of rARU, elicited 437 μg CDTAantibody/ml (112.0 μg Ab/μg rARU injected) compared to 518 μg CDTAantibody/ml for SF-rARUsucc (34.9 μg Ab/μg rARUsucc injected). Althoughthe specific immunogenic activity for the rARUsucc was lower than thatof the rARU in the SF conjugates, there was no statistical differencebetween the levels of CDTA antibody elicited by the two conjugates (437μg Ab/ml for SF-rARUsucc vs 242 μg Ab/ml for SF-rARU).

[0056] K1-rARUsucc, that elicited 390 μg CDTA antibody/ml, hadcomparable specific immunogenic activity of its rARU component (48 μgAb/ml per μg rARUsucc).

Example 6

[0057] CDTA Neutralizing Antibodies.

[0058] Individual sera obtained 7 days after the third injection of theconjugates were assayed individually for their neutralization ofapproximately 200 times the cytotoxic dose of CDTA on human intestinalepithelial HT-29 cells. All sera from the mice immunized with theconjugates had a neutralizing titer greater than or equal to 64. Thegeometric mean and range of neutralizing titers for each conjugate isshown in Table 2. TABLE 2 Serum neutralizing activity against the invitro cytotocicity for HT-29 cells of Clostridium difficile toxin A(CDTA) Reciprocol μg Ab/ml neutra;ization titer Immunogen (ELISA) (GMand range) Pn14-rARU 194 104 64-256 Pn14-rARU succ 371 111 64-128SF-rARU 613 194 64-256

Example 7

[0059] Protection against lethal challenge with CDTA (Table 3).

[0060] Hsd/ICR mice were injected with SF-rARU, SF-rARUsucc or rARU asdescribed in EXAMPLE 4 above. One week after the third injection, themice were challenged intraperitoneally with a lethal dose (150 ng) ofCDTA. Almost all mice vaccinated with either conjugate or rARU wereprotected. Based upon the amount of rARU injected, rARU and SF-rARUelicited similar levels of anti-CDTA. As expected, SF-rARUsucc elicitedlower levels of anti-CDTA than the other two immunogens but therecipients were comparably protected.

[0061] Conjugate-induced antibody levels approached or surpassed theneutralizing activity of an affinity-purified goat antibody, containing0.5 mg/ml, that was raised against formalin inactivated CDTA. TABLE 3Protection of mice against lethal challenge with 150 ng of Clostridiumdifficile toxin A (CDTA)^(a) induced by vaccination withpolysaccharide-rARU conjugates CDTA Reciprocal μg rARU Survivals/antibodies neutralization Immunogen injected total (ELISA)^(b) titer^(c)rARU 6.94 19/20 717 (621-863) 128-256 SF-rARU 3.90 17/20 437 (372-547)128-256 SF-rARUsucc 6.94 19/20 242 (172-443)  64-256 PBS 0  2/15 Notdetermined <2

[0062] This invention has been described by a direct description and byexamples. As noted above, the examples are meant to be only examples andnot to limit the invention in any meaningful way. Additionally, onehaving ordinary skill in the art to which this invention pertains inreviewing the specification and claims which follow would appreciatethat there are equivalents to those claimed aspects of the invention.The inventors intend to encompass those equivalents within thereasonable scope of the claimed invention.

LITERATURE CITED

[0063] U.S. Pat. No. 5,098,826 (Wilkins et al.) (1992).

[0064] U.S. Pat. No. 5,736,139 (Kink et al.) (1998)

[0065] U.S. Pat. No. 5,919,463 (Thomas et al.) (1999)

[0066] Lyerly, D. M. and T. D. Wilkins, in Infections of theGastrointestinal Tract, Chapter 58, pages 867-891, Raven Press, Ltd, NewYork 1995

[0067] Moncrief et al., Infect. Immun. 65:1105-1108 (1997);

[0068] Barroso et al., Nucl. Acids Res. 18:4004 (1990);

[0069] Dove et al. Infect. Immun. 58:480-488 (1990)). (

[0070] Krivan et al., Infect. Immun. 53:573-581 (1986);

[0071] Tucker, K. and T. D. Wilkins, Infect. Immun. 59:73-78 (1991)).

[0072] Just et al. Nature 375:500-503 (1995),

[0073] Just et al. J. Biol. Chem 270:13932-13939 (1995)).

[0074] Hofmann et al J. Biol. Chem. 272:1 1074-11078 (1997),

[0075] Faust and Song, Biochem. Biophys. Res. Commun. 251:100-105(1998))

[0076] Lyerly et al. Current Microbiology 21:29-32 (1990)

1. An immunogenic composition comprising a recombinant proteincomponent, wherein said protein component comprises at least onerepeating unit of C. difficile toxin A (rARU) or at least one repeatingunit of C. difficile toxin B (rBRU) or at least one repeating unit ofeach toxin A and toxin B.
 2. The immunogenic composition of claim 1wherein said recombinant protein component comprises at least two ofsaid repeating units that are genetically fused to each other.
 3. Theimmunogenic composition of claim 1 wherein said recombinant proteincomponent comprises at lest two of said repeating units that arechemically bound to each other.
 4. The immunogenic composition of claim1, wherein said composition elicits antibodies that neutralize toxin Aor toxin B or both.
 5. The immunogenic composition of claim 1 thatelicits a protective response in a mammalian host against strains of C.difficile.
 6. The immunogenic composition of claim 2 that elicitsantibodies that neutralize toxin A or toxin B or both.
 7. Theimmunogenic composition of claim 2 that elicits a protective response ina mammalian host against strains of C. difficile.
 8. The immunogeniccomposition of claim 3 that elicits antibodies that neutralize toxin Aor toxin B or both.
 9. The immunogenic composition of claim 3 thatelicits a protective response in a mammalian host against strains of C.difficile.
 10. A pharmaceutical composition comprising the immunogeniccomposition of any one of claims 1-9 in a pharmaceutically acceptablecarrier.
 11. A method of conferring a protective response in a mammalianhost comprising administering a therapeutically effective amount of theimmunogenic composition of any one of claims 1-9 to a mammalian host.12. A method of conferring a protective response in a mammalian hostcomprising administering a therapeutically effective amount of thepharmaceutical composition of claim 10 to a mammalian host.
 13. Theimmonogenic composition of claim 1, wherein said protein component istoxin A or a fragment threreof.
 14. The immunogenic composition of claim13, wherein said protein component comprises a recombinant amino acidsequence that comprises the toxin A repeating units (rARU) or a fragmentthereof.
 15. The immunogenic composition of claim 14, wherein saidprotein is a fusion protein.
 16. The immonogenic composition of claim 1,wherein said protein component is toxin B or a fragment threreof. 17.The immunogenic composistion of claim 16, wherein said protein comprisesa recombinant amino acid sequence that comprises the toxin B repeatingunits (rBRU) or a fragment thereof.
 18. The immunogenic composition ofclaim 17, wherein said protein is a fusion protein.
 19. The immunogeniccomposition of claim 1, wherein said immunogenic composition elicits ina mammalian host an immune response that is T-cell dependent.
 20. Theimmunogenic composition of claim 1, wherein said immunogenic compositionelicits in a mammalian host an immune response that is T-cellindependent.
 21. The immunogenic composition of claim 1, wherein saidimmunogenic composition elicits in a mammalian host an immune responsethat is both T-cell dependent and T-cell independent.
 22. Theimmunogenic composition of claim 19 or 20 or 21, wherein said immuneresponse is a cellular dependent immune response.
 23. The immunogeniccomposition of claim 19 or 20 or 21, wherein said immune responseresults in a booster effect in said mammalian host.
 24. The immunogeniccomposition of claim 19 or 20 or 21, wherein said immune responseelicits a protective response to a strain of C. difficile.
 25. Theimmunogenic composition of claim 19 or 20 or 21, wherein saidimmunogenic composition elicits a humoral immune response in a mammalianhost.
 26. The immunogenic composition of claim 19 or 20 or 21, whereinsaid immunogenic composition elicits both a humoral immune response anda cellular dependent immune response in a mammalian host.
 27. Theimmunogenic composition of claim 19 or 20 or 21, wherein said immuneresponse elicits a protective response to a strain of a C. difficile.28. An immunogenic composition comprising a recombinant proteincomponent, wherein said protein component comprises a recombinant aminoacid sequence that comprises the Clostridium difficile toxin A repeatingunits (rARU) or a fragment thereof and wherein said composition furthercomprises a pharmaceutically acceptable carrier.
 29. An immunogeniccomposition comprising a recombinant protein component, wherein saidprotein component comprises a recombinant amino acid sequence thatcomprises the Clostridium difficile toxin B repeating units (rBRU) or afragment thereof and wherein said composition further comprises apharmaceutically acceptable carrier.
 30. An immunogenic compositioncomprising a recombinant protein component, wherein said proteincomponent comprises a recombinant amino acid sequence that comprises theClostridium difficile toxin A repeating units (rARU) or the Clostridiumdifficile toxin B repeating units (rBRU) or a fragment of either thereofand wherein said composition further comprises a pharmaceuticallyacceptable carrier.
 31. An immunogenic composition comprising arecombinant protein component, wherein said protein component comprisesa recombinant amino acid sequence that comprises the Clostridiumdifficile toxin A repeating units (rARU) or a fragment of thereof andthe Clostridium difficile toxin B repeating units (rBRU) or a fragmentthereof and wherein said composition further comprises apharmaceutically acceptable carrier.
 32. A vaccine comprising theimmunogenic composition of any one of claims 28, 29, 30 or
 31. 33. Thevaccine of claim 32, wherein said vaccine is formulated for use inhumans.
 34. The vaccine of claim 32, wherein said vaccine is formulatedfor use in animals.
 35. A method for producing an immunogeniccomposition, comprising constructing a genetic sequence encoding arecombinant protein component, wherein said genetic sequence is isolatedfrom a strain of Clostridium difficile; expressing said recombinantprotein in a microbial host; recovering said recombinant protein from aculture of said host; and recovering said recombinant protein component;and wherein said protein component is Clostridium difficile toxin Arepeating unit (rARU) or fragement thereof or is Clostridium difficiletoxin B repeating unit (rBRU) or fragement thereof.
 36. The method ofclaim 35, wherein the expression of said genetic sequence is regulatedby an inducible promoter operatively positioned upstream of saidsequence and functional in said host.
 37. The method of claim 36,wherein said microbial host is Escherichia coli.
 38. The method of claim37, wherein the recombinant protein is expressed at a level greater thanabout 10 mg/ml.
 39. The method of claim 37, wherein the recombinantprotein is expressed at a level greater than about 50 mg/liter of saidculture.
 40. The method of claim 37, wherein the recombinant protein isexpressed at a level greater than about 100 mg/liter of said culture.41. The method of claim 37, wherein said protein is greater than about50 kDa.
 42. The method of claim 37, wherein said protein is greater thanabout 90 kDa.
 43. The method of claim 37, wherein said protein isrecovered by ammonium sulfate precipitation followed by ion exchangechromatography.
 44. The method of claim 37, wherein said protein issuccinylated.
 45. A recombinant genetic sequence comprising at least onegene encoding a protein component from a strain of Clostridiumdifficile.
 46. The recombinant sequence of claim 45, wherein said geneencodes toxin A or a fragment thereof.
 47. The recombinant sequence ofclaim 46, wherein said gene encodes the toxin A repeating units (rARU)or a fragment thereof.
 48. The recombinant sequence of claim 45, whereinsaid gene encodes toxin B or a fragment thereof.
 49. The recombinantsequence of claim 48, wherein said gene encodes the toxin B repeatingunits (rBRU) or a fragment thereof.
 50. The recombinant sequence ofclaim 45, comprising a first gene encoding toxin A or a fragment thereofand a second gene encoding toxin B or fragement thereof.
 51. Therecombinant sequence of claim 50, wherein said first gene encodes thetoxin A repeating units (rARU) or a fragment thereof and said secondgene encodes the toxin B repeating units (rBRU) or a fragment thereof.52. An expression vector comprising the genetic sequence of claim 45 orclaim 50 and a gene that confers a selective phenotype upon a microbialhost.
 53. The expression vector of claim 52, wherein said selectivephenotype is resistance to kanamycin.
 54. A microbial host transformedwith the expression vector of claim 52 or claim
 53. 55. The use of theimmunogenic composition of any one of claims 1-9 and 13-31 for theproduction of antibodies for passive immune therapy against a strain ofsaid pathogenic microorganism.